Accurate formation of the retinotopic map is critical for our ability to see. During this process, retinal axons are guided to form a precise topographic map of connections in the brain that allow visual space to be perceived. The molecular basis of these guidance mechanisms is not yet known. The long term objective of this proposal is to understand how repulsive cues act to guide axons to form the retinotopic map. The recent discovery of a variety of repulsive cues act to guide axons to form the retinotopic map. The recent discovery of a variety of repulsive cues has modified our understanding of how axons re guided. It is timely to address how these cues function in vivo. Repulsive guidance molecule (RGM) and ephrin-A5 are expressed in high posterior to low anterior gradients in the tectum of lower vertebrates during the formation of the retinotectal projection. Current advances have suggested specific hypotheses of how these two cues act in vivo to shape the retinotectal map: 1) RGM guides initial retinotectal axon outgrowth. 2) Ephrin-A5 prevents retinal axons from exiting the tectum and modulates lateral branching after initial axon outgrowth is completed. The proposed experiments will test these hypotheses. The changes in retinotectal projects caused by perturbing RGM and ephrin-A5 in vivo will be observed. Chick and zebrafish embryos will be studied; they are well characterized and each offers distinct advantages to achieve our goals. Acute and local inactivation of RGM and ephrin-A5 during retinotectal map formation will be achieved by chromophore-assisted laser inactivation (CALI) during initial axon outgrowth and later during lateral branching. Infection with recombinant retrovirus will generate chronic misexpression of ephrin-A5 in the chick tectum. The changes in retinotectal projects will be assessed by high resolution axon tracing and live imaging. The combination of these two approaches will provide strong complementary information that will test the proposed hypotheses. As RGM and ephrin-A5 are likely used for axon guidance and formation of topographic order, the proposed experiments are of significant clinical relevance.